The Role of the Seagrass Leaf Microbiome in Assisting Nitrogen Uptake by the Western Australian Seagrass, Posidonia Sinuosa
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Edith Cowan University Research Online Theses: Doctorates and Masters Theses 2017 The role of the seagrass leaf microbiome in assisting nitrogen uptake by the Western Australian seagrass, Posidonia sinuosa Flavia Tarquinio Edith Cowan University Follow this and additional works at: https://ro.ecu.edu.au/theses Part of the Ecology and Evolutionary Biology Commons Recommended Citation Tarquinio, F. (2017). The role of the seagrass leaf microbiome in assisting nitrogen uptake by the Western Australian seagrass, Posidonia sinuosa. https://ro.ecu.edu.au/theses/2046 This Thesis is posted at Research Online. https://ro.ecu.edu.au/theses/2046 Edith Cowan University Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorize you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. 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The role of the seagrass leaf microbiome in assisting nitrogen uptake by the Western Australian seagrass, Posidonia sinuosa Flavia Tarquinio M.Sc. in Marine Biology Polytechnic University of Marche This thesis is presented for the degree of Doctor of Philosophy School of Science Edith Cowan University 2017 USE OF THESIS The Use of Thesis statement is not included in this version of the thesis. ABSTRACT Microorganisms play a key role in facilitating the cycling of several elements in coastal environments, including nitrogen (N). N is a key component for maintaining high seagrass productivity and is often the limiting nutrient in marine environments. Seagrasses harbour an abundant and diverse microbial community (the ‘microbiome’), however their ecological and functional roles related to the seagrass host are still poorly understood, in particular regarding N cycling. Microorganisms capable of mineralising dissolved organic nitrogen (DON) may play a pivotal role in enhancing N availability in coastal environments such as seagrass meadows. Thus, the overall aim of my thesis was to enhance current understanding of abundance and diversity of the microbial community associated with seagrass meadows and their ecological role, with specific focus on N cycling. This was achieved by using molecular techniques together with 15N-enrichment experiments and nanoscale imaging techniques. Firstly, I reviewed the literature on the potential effects that microorganisms associated with both the above- and belowground seagrass tissue may have on plant fitness and the relevance of the seagrass microbiome and I have highlighted literature gaps. For my second chapter, I determined the abundance and community composition of bacteria and archaea associated with seagrass Posidonia sinuosa meadows in Marmion Marine Park, southwestern Australia. Data were collected from different seagrass meadows and meadow ‘microenvironments’, i.e. seagrass leaf surface, sediment and water column. I performed the quantitative polymerase chain reaction (q-PCR) targeting a series of bacterial and archaeal genes: 16S rRNA, ammonia oxidation genes (amoA) and genes involved in mineralisation of DON, via the urease enzyme (ureC). High-throughput sequencing was applied to 16S rRNA and amoA genes, to explore the diversity of these microbial assemblages related to P. sinuosa meadow microenvironments. Results from this chapter show that the P. II sinuosa leaf biofilm represents a favourable habitat for microorganisms, as it hosts a significantly higher microbial abundance compared to the sediment and water. Moreover, 16S rRNA and amoA sequencing data indicate a high degree of compartmentalisation of functional microbial communities between the microenvironments of the seagrass meadow (leaf, sediment and water column), pointing towards the existence of a core seagrass leaf microbiome that could have specific interactions with the plant. For my third chapter I determined the role that microorganisms inhabiting P. sinuosa seagrass leaves may play in the recycling of DON, and subsequent transfer of inorganic N (DIN) into plant tissues. To achieve this, I performed an experiment whereby seagrass leaves with and without microorganisms were incubated with DO15N, and I traced the fine-scale uptake and assimilation of microbially processed N into seagrass cells, using nanoscale secondary ion mass spectrometry (NanoSIMS). Results from this chapter show for the first time that seagrass leaf epiphytic microorganisms facilitated the uptake of 15N from DON, which was unavailable to the plant in the absence of epiphytes. This indicates that seagrass leaves have limited to no ability to take up DON, and the seagrass leaf microbiome could therefore play a much more significant role than previously thought in enhancing plant health and productivity. Finally, I determined the net nitrification rates associated with ammonia- oxidising microorganisms (AOM) inhabiting P. sinuosa leaf surfaces, and explored whether AOM facilitated, or competed for, the plant’s N uptake. My findings show + that AOM may compete with seagrasses for NH4 uptake, but that their potential to outcompete seagrass epiphytic algae for DIN uptake indicates that AOM on seagrass leaves may serve as a ‘biocontrol’ over excess epiphytic algal growth. In summary, the present thesis represents a significant advance in our understanding of the seagrass leaf-microbiome relationship and transformations of N within seagrass meadows. Moreover, it opens up new questions for future research not only on seagrass-microbiome interactions but other macrophytes in aquatic systems that may benefit from the presence of specific N-cycling microorganisms. III IV DECLARATION I certify that this thesis does not, to the best of my knowledge and belief: incorporate without acknowledgment, any material previously submitted for a degree or diploma in any institution of higher education; contain any material previously published or written by another person except where due reference is made in the text of this thesis; contain any defamatory material; or contain data that have not been collected in a manner consistent with ethics approval. Flavia Tarquinio August 2017 V This Thesis is Dedicated to My Family VI ACKNOWLEDGMENTS There are many people that helped me through this journey and that without them I would not have completed this thesis. First, I would like to thank my family for all the support. So probably noone will get offended if I write a couple of sentences in Italian! Grazie mille per tutto quello che avete fatto e farete per me, per sostenermi anche se siete lontani e per l’amore che continuo a ricevere, mi sembrate cosi vicini!!!Grazie a mia mamma mio padre e mio fratello vi voglio bene! Anche alle nonne, cosi non si offende nessuno! Now, a special thank to all my supervisors who have been working like crazy during the past weeks to allow me to get this work of art out in time! SO really thanks to all of you: Glenn Hyndes, Annette Koenders, Bonnie Laverock and Christin Sawstrom. You guys have been fantastic : ) A special thanks to all the researchers who also supported me during these years. Firstly, Megan Huggett, you did not just open your knowledge to me but also your home, so thanks : ) Thanks to Olly Berry who hosted me for a year at CSIRO in his fantastic lab and Jeremy Bougoure who gave me a free week run at the NasoSIMS (but don t tell anyone!) and was instrumental in helping me with Chapter 4. Thanks to Andrea Paparini who helped me through the sequencing at Murdoch University, but also for being there for my desperate calls. Also, thanks to Paul Guagliardo from the CMCA at UWA. A HUGE THANKS to La babba and Annina, friends before being colleagues, and thanks Duccio for feeding me in the last nights before thesis submission. Thanks to all of you my friends, in Italy (Lucy, Ros, Ca’ and Vale) and here (Ana, Chris, Bruno, Fede , Aldo, Giulia, all the CMER group) especially my good friend Titi, and sorry if I am not that precise/good, but I am going to send this thesis out in 15 so see you soon…….for a big party of course : D VII LIST OF PUBLICATIONS INCLUDED AS PART OF THE THESIS This thesis is presented as a series of manuscripts that are to be submitted for publication in international journals. I am the first author with a contribution of more than 50% to each publication. The thesis conforms to the “thesis with publication” style of Edith Cowan University. VIII IX Contents Use of thesis .................................................................................................................. i Declaration ................................................................................................................... v Acknowledgments.....................................................................................................